Milliplex map bovine cytokine magnetic bead panel assay kits

Arterial plasma was collected following ventilation (0 [pre-ventilation], 15 min [end of ventilation] and 1, 3, 6, 12 and 24 h [post-ventilation recovery]) and used to quantify levels of IFNγ, IL-1β, IL-10, TNFα, IL-8 and IL-6 using a Milliplex MAP bovine cytokine magnetic bead panel assay kits according to manufacturer’s instructions (cat#: CYT1-91 K; MerckMillipore, USA). In brief, 96-well plates were coated with samples, assay buffer, serum matrix and antibody-immobilised beads. Plates were incubated overnight at 4 °C, washed and incubated with detection antibodies for 1 h. Streptavidin-phycoerythrin was added to the plates for 30 min. Sheath fluid was added to the plates and cytokine concentrations were quantified using a Bio-Plex MAGPIX® Multiplex reader with xPOTENT® software (Bio-Rad, CA, USA).

CSF samples collected at post-mortem were assessed for proteins IL-1β, IL-6, IL-8. IL-10, tumor necrosis factor (TNF) and interferon γ-produced protein (IP-10) using Milliplex MAP bovine cytokine magnetic bead panel assay kits (cat#: BCYT1-33K; MerckMillipore, Burlington, MA, USA) as previously described (15 (link)). In brief, 96 well plates were first washed and coated in sample, assay buffer, serum matrix, and antibody-immobilised beads. The plates were incubated overnight at 4°C. Following overnight incubation, the plates were washed and incubated with the detection antibodies for 1 h. Streptavidin-phycoerythrin was added to the plates for 30 min at room temperature. Sheath fluid was then added to each of the plates. Protein concentrations were quantified using a Bio-Plex MAGPIX® Multiplex reader with xPOTENT® software (Bio-Rad, CA, USA). Internal quality controls were included in each assay kit. Cytokine levels were within detection limit in all samples. Standards were bovine recombinant IL-1 β (range, 12.8–200,000 pg/ml; assay sensitivity, 3.9 pg/ml), IL-6 (range, 7.7–120,000 pg/ml; assay sensitivity, 0.65 pg/ml), IL-8 (range, 0.77–12,000 pg/ml; assay sensitivity, 1.62 pg/ml), IL-10 (range. 1.6–25,000 pg/ml; assay sensitivity, 0.86 pg/ml), TNF (range, 48–750,000 pg/ml; assay sensitivity, 9.34 pg/ml), and IP-10 (range, 0.77–12,000 pg/ml; assay sensitivity, 0.46 pg/ml).